Computational protocol: Living biointerfaces based on non-pathogenic bacteria to direct cell differentiation

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[…] For bacterial viability studies, after 1, 2 or 4 days, biofilms were gently washed twice with sterile NaCl 0.85% w/v solution and incubated for 30 min using the BacLight LIVE/DEAD kit (Life Technologies), containing SYTO9 (5 µM) and propidium iodide (30 µM) in NaCl 0.85% w/v. Afterwards, samples were washed and mounted using BacLight Oil mounting medium (Life Technologies). Bacteria were counted using Fiji – ImageJ software, using at least four microscope fields at 40× magnification for every sample. The viability was determined as the ratio between the viable and total number of bacteria.For metabolism analysis, biofilms were prepared following the biofilm formation protocol. Afterwards, the medium was changed to either DMEM or DMEM containing tetracycline (TC, 10 µg/mL)) or DMEM containing 1% penicillin-streptomycin (P/S). Biofilms were kept at 37°C in a humidified atmosphere under 5% CO2, and the pH was monitored as a function of time (0, 0.5, 1, 1.5, 2, 3, 4, 5, 24 and 48 h) using a pH-meter (Eutech Instruments). [...] Cultures were fixed at 4°C with 70% ethanol, 37% formaldehyde and glacial acetic acid (20:2:1) for 15 min and then blocked with 5% goat serum in PBS++ for 1 h at RT. The samples were incubated with MF-20 antibody against sarcomeric myosin (Developmental Studies Hybridoma Bank, University of Iowa, USA) at 1:250 dilution in PBS++ and 5% goat serum, followed by two washes with PBS++/Tween 20 (0.5% v/v) and another blocking step with 5% goat serum in PBS++ for 10 min at RT. Then were washed twice with PBS++/Tween 20 (0.5% v/v) and incubated with rabbit anti-mouse Cy3-conjugated secondary antibody (Jackson Immunoresearch) at 1:200 dilution in PBS++ with 5% goat serum. Samples were then washed three times with PBS++/Tween 20 (0.5% v/v), mounted with Vectashield-DAPI, and observed under an epifluorescence microscope (Nikon Eclipse 80i).Images from the fluorescence microscope (DAPI channel - nuclei, and Cy3 channel - sarcomeric myosin) of the C2C12 culture were acquired at 10× magnification, transformed to an 8-bit grayscale bitmap (Fiji - ImageJ software) and segmented using the Trainable Weka Segmentation plugin to create a binary mask, for both DAPI and Cy3 channels. Total nuclei per image were counted using the particle analysis command. Then, the segmented DAPI channel image was subtracted from the Cy3 channel segmented image, and the remaining nuclei were counted and assigned to non-differentiated cells. The fraction of differentiated cells was calculated subtracting the non-differentiated nuclei from the total nuclei count.For vinculin and actin visualisation, a slightly different protocol was used. Cell fixation was performed using formalin for 30 min at 4°C. Then cells were blocked with 1% BSA in PBS++. A monoclonal anti-vinculin antibody (Sigma), diluted 1:400 in BSA 1%/PBS++ was used as primary antibody. The incubation lasted 1 hour at RT. Then, cells were washed twice with PBS++/Tween 20 (0.5% v/v), and incubated with rabbit anti-mouse Cy3 conjugated (Jackson Immunoresearch), 1:200 dilution, with FITC-conjugated phallacidin (Life Technologies) diluted 1:100 in 1% BSA/PBS++/Tween 20 (0.5% v/v). The secondary antibody was incubated 1 hour at RT in absence of light. Cells were washed three times with PBS/Tween 20 (0.5% v/v) and mounted with Vectashield-DAPI (Vector Laboratories). […]

Pipeline specifications

Software tools ImageJ, TWS
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Lactococcus lactis, Homo sapiens, Lactobacillus delbrueckii subsp. lactis